Single-telomere-specific (TTAGGG)n tract lengths and instabilities cannot be measured globally using current methods; the ability to do so would be a major, transformative addition to the arsenal of tools for analyzing critical telomere loss an telomere elongation events in cancer. The shortest telomere or a small subset of the shortest telomeres in a cell will determine the onset of senescence, apoptosis, or genome instability; single (TTAGGG)n tracts are crucial for the function of telomeres and the biological effects of telomere attrition and dysfunction. Telomere loss, breakage, fusion, and rejoining are highly elevated in cancer, but current methods for detecting and measuring these mutational events at the molecular level are limited and low-throughput. The technology we propose to develop here would permit quantitative, single- allele-resolution measurements of telomere length and instability, enabling unprecedented insights into the role(s) telomere loss, telomere breakage/re-joining, and telomerase or ALT dependent telomere elongation play in carcinogenesis, including mechanistic insights into molecular events mediating these processes and translational insights for the potential prognostic and tumor stratification applicability of the methods. In this method, input genomic DNA is labeled with fluorescent dyes specific for (TTAGGG)n sequences and for linked subtelomeric DNA. The labeled individual DNA fragments are linearized (stretched) and imaged in the nano-channels of Bionano Genomics system at very high throughput. The lengths of the telomere are measured accurately and the distances between probes in the subtelomere region are determined accurately to infer the identity of the telomere. Our goals for this R21 study are to establish feasibility for (1) high-throughput single-molecule detection and quantitation of (TTAGGG)n tracts in genomic DNA samples and (2) subtelomere probe development and efficient co-labeling of telomeres and subtelomeres in the context of total genomic DNA, including conversion of the labeled DNA to double- stranded DNA suitable for nano-channel analysis; and (3) proof-of-principle results for the technology and its applicability for cancer research using data generated and analyzed for a normal and a cancer cell line. There are significant technical challenges inherent in this early-stage technology development project, but the extraordinary payoff for cancer research will be a high-throughput ability to probe mechanisms of telomere length regulation and telomere mutation at single-telomere resolution, in small (ultimately single-cell) samples of both dividing and non-dividing cells.